This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. ABSTRACT: The realization that mitochondria take up Ca2+ under normal physiological conditions, and contribute to Ca2+ homeostasis and signaling (Rizzuto et al., 1998, 2003;Csordas et al., 1999), has led to the hypothesis of special micro-domains existing between the mitochondrial surface and the endoplasmic reticulum (ER). These microdomains are thought to be sites where IP3-receptors (IP3-R) are present in high concentration on ER membranes in close proximity to the mitochondrial OM. Likewise, in the same regions, there may be locally high concentrations of OM and IM proteins involved in mitochondrial Ca2+ transport, such as VDAC. Rizzuto et al. (1998), Csordas and Hajnoczky (2001), and others have used confocal microscopy to demonstrate that there are numerous regions in mammalian cells where mitochondria and ER are co-localized. In a recent review, Rizzuto et al. (2003) raise the question: Is proximity of mitochondria to Ca2+ channels on the ER stochastic in nature (a consequence of the extensive reticulation of the two membrane systems) or does a specific mechanism ensure co-localization? In tomographic reconstructions of isolated rat-liver mitochondria, linear "tethers" (10-50 nm in length) appear to attach small vesicles to the mitochondrial OM. Light microscopic immuno-labeling experiments with anti-IP3-R indicate that a sub-set of these mitochondria have ER attached which can be released by protease. (Corresponding EM immunolabelling experiments are planned.) In recent reconstructions of both plastic-embedded and cryo-sectioned rat liver, numerous OM-ER tethers have been observed, many of which originate on ER-bound ribosomes. These observations suggest that the tether protein may attach to the mitochondrial surface while being synthesized on ER-bound ribosomes (i.e., before release from the ribosome). We are hypothesizing that the tethers observed in electron tomographic reconstructions of isolated mitochondria (connecting the OM to putative ER vesicles), and of mitochondria in situ are comprised of proteins whose function is to ensure that a fraction of mitochondria are always in close proximity (10-50 nm) to the ER. Accordingly, (1) the primary aim of this project is to characterize the mitochondrial-ER tethers in liver and in mammalian cell lines in which knock-out or knock-down techniques can be used to alter normal expression of proteins that might be involved in the association of mitochondria and ER. These include IP3-receptor (mutant available in Hajnoczky laboratory), VDAC (knockouts available from several collaborators, including K. Kinnally), and other Ca2+ transport proteins. Candidate proteins will be screened by EM immunolabeling of isolated rat liver mitochondria containing attached ER vesicles: proteins whose antibodies bind to the vicinity of the tethers will be selected for in situ imaging experiments. A question raised by the above observations is whether the formation of tethers is dependent on continuous protein synthesis (i.e., the tethers are transient, perhaps a consequence of import of a particular protein into mitochondria) or if the connections are stable. The observation of numerous tethers on isolated mitochondria supports but does not prove the second scenario. Therefore, (2) a second aim is to determine whether inhibition of protein synthesis (by cyclohexamide or other inhibitors) effects tether length or density. A third (and somewhat unrelated) aim is (3) to use the motif search algorithm to determine the orientation of the ribosomes (ER-bound and free) in close proximity to the mitochondrial outer membrane. There is considerable controversy regarding the occurrence of vectorial translational of some mitochondrial proteins occurs, i.e., whether certain precursor proteins are imported into mitochondria while they are being synthesized on ribosomes. Ribosomes on the ER are presumed to have a standard orientation (i.e., large subunit toward the translocon receptor). We have found in tomograms of mitochondria-ER micro-domains that up to one-third of the ribosomes in the space between the OM and ER are not attached to the ER, and some of these appear to be very close to the mitochondrial OM. If the ribosomes in close proximity to the OM are oriented with large subunit towards the OM, this would be a strong indication that vectorial translation of mitochondrial proteins was taking place. To succeed, all three objectives require progress under RVBC technological development projects associated with TRD#1: use of native tissue and cells, high resolution tomography, and a sensitive motif searching algorithm. References 1. Csordas, G., Thomas, A. P., and Hajnoczky, G. (1999). Quasi-synaptic calcium signal transduction between endoplasmic reticulum and mitochondria. EMBO J 18:96-108. 2. Csordas, G. and Hajnoczky, G. (2001). Sorting of calcium signals at the junctions of endoplasmic reticulum and mitochondria. Cell Calcium 29:249-262. 3. Rizzuto, R., Brini, M., Murgia, M., and Pozzan, T. (1998). Microdomains with high Ca2+ close to IP3-sensitve channels that are sensed by neighboring mitochondria. Science 262:744-747. 4. Rizzuto, R., Duchen, M. R., and Pozzan, T. (2004). Flirting in little space: The ER/mitochondrial Ca2+ liaison. Science's STKE re1:1-9. The paper characterizing the physical connections (tethers) between the mitochondrial outer membrane (OM) and endoplasmic reticulum (ER) and their influence on calcium regulation in mammalian cells (Csordas et al., J. Cell Biology 2006) has generated considerable interest in the calcium field. The next phase of the project is to determine the length and spatial distribution of the OM-ER tethers in intact frozen-hydrated cells under different physiological conditions and in mutants lacking candidate components of the tethers. In the previous reporting periods, extensive work with visualization of tethers was done, and the results were reported at several meetings. To add to this work, we received a plastic-embedded block of RBL cells from the Hajnoczky lab. A double-tilt tomographic reconstruction was made using the HVEM, and mitochondria were selected for future tomograms to investigate differences in cristae between the cell types so far used in this study.